Cho E J, Bae J B, Kang J G, Roe J H
Department of Microbiology, College of Natural Sciences, Seoul National University, Korea.
Nucleic Acids Res. 1996 Nov 15;24(22):4565-71. doi: 10.1093/nar/24.22.4565.
The rpoA gene, encoding the alpha subunit of RNA polymerase, was cloned from Streptomyces coelicolor A3(2). It is preceded by rpsK and followed by rplQ, encoding ribosomal proteins S11 and L17, respectively, similar to the gene order in Bacillus subtilis. The rpoA gene specifies a protein of 339 amino acids with deduced molecular mass of 36,510 Da, exhibiting 64.3 and 70.7% similarity over its entire length to Escherichia coli and B. subtilis alpha subunits, respectively. Using T7 expression system, we overexpressed the S. coelicolor alpha protein in E. coli. A small fraction of this protein was found to be assembled into E. coli RNA polymerase. Antibody against S. coelicolor alpha protein crossreacted with that of B. subtilis more than with the E. coli alpha subunit. The ability of recombinant alpha protein to assemble beta and beta' subunits into core enzyme in vitro was examined by measuring the core enzyme activity. Maximal reconstitution was obtained at alpha2:beta+beta' ratio of 1:2.3, indicating that the recombinant alpha protein is fully functional for subunit assembly. Similar results were also obtained for natural alpha protein. Limited proteolysis with endoproteinase Glu-C revealed that S. coelicolor alpha contains a tightly folded N-terminal domain and the C-terminal region is more protease-sensitive than that of E. coli alpha.
编码RNA聚合酶α亚基的rpoA基因是从天蓝色链霉菌A3(2)中克隆得到的。它位于rpsK基因之后，rplQ基因之前，rpsK和rplQ分别编码核糖体蛋白S11和L17，这与枯草芽孢杆菌中的基因顺序相似。rpoA基因编码一个由339个氨基酸组成的蛋白质，推导的分子量为36,510 Da，其全长与大肠杆菌和枯草芽孢杆菌的α亚基分别具有64.3%和70.7%的相似性。利用T7表达系统，我们在大肠杆菌中过表达了天蓝色链霉菌的α蛋白。发现一小部分这种蛋白组装进了大肠杆菌RNA聚合酶中。抗天蓝色链霉菌α蛋白的抗体与枯草芽孢杆菌的α蛋白发生交叉反应的程度高于与大肠杆菌α亚基的交叉反应。通过测量核心酶活性，检测了重组α蛋白在体外将β和β'亚基组装成核心酶的能力。在α2:β+β'比例为1:2.3时获得最大重组，表明重组α蛋白在亚基组装方面完全有功能。天然α蛋白也得到了类似结果。用内肽酶Glu-C进行的有限蛋白酶解表明，天蓝色链霉菌α蛋白含有一个紧密折叠的N端结构域，其C端区域比大肠杆菌α蛋白的C端区域对蛋白酶更敏感。
